The present invention relates to a novel method for the analysis of human-derived samples that can provide information useful in the therapy of osteoporosis. Specifically, the present invention relates to a method for the analysis of genetic polymorphism of genome DNA in human-derived samples in order to anticipate which is the most effective remedy for osteoporosis, in particular, among vitamin D, estrogen, and vitamin K2. Also, the present invention relates to a method for anticipating, based on a combination of genetic polymorphisms of genome DNA in a human-derived sample, that the sample is derived from an individual who shows specified priority in sensitivity to the above medicines. Further, the present invention relates to a kit for the analysis of genetic polymorphisms that can be used in the above method.
Osteoporosis is the state of a disease in which bone mass (the amount of minerals, mainly calcium contained in bone) decreases and the fine structure of bone tissue changes so that the bone becomes brittle and tends to be broken. It occurs mostly in females after menopause and in senior males. It is said that the number of patients with osteoporosis is presumably 10,000,000 in Japan. It is anticipated that as the ratio of elderly persons in the population increases, the number of patients will henceforth inevitably increase.
At present, various medicines such as bone activators, e.g., calcium preparations, vitamin D, etc., bone resorption depressants, e.g., estrogen, etc., and osteogenesis accelerators, e.g., vitamin K, etc., are used to treat osteoporosis. However, their therapeutical effects vary randomly depending on the patient and there have been made almost no study as to how to use a right medicine with a right patient. Since it has been taken as a rule that these remedies are administered singly, there is currently no way other than actually administering a single medicine to patients for several years and obtaining results before it can be judged based on the results which medicine is most effective. This is extremely inefficient.
On the other hand, recent studies on genes have suggested a relationship between some genetic polymorphisms and the sensitivity of a patient to remedies for osteoporosis. For example, there is a report that the genotype A that is not cleaved with Apa I in the intron region between exon 8 and exon 9 of the vitamin D receptor (hereafter, referred to as VDR) gene is more sensitive to vitamin D than the genotype a that is cleaved with the same restriction enzyme [see, for example, JP-A-8-126497 and JP-A-8-126500].
Also, Shiraki et al. [Resume of 1997 Conference of Japanese Society for Bone and Mineral Research, page 52] describe that in accordance with the results of examinations on the relationships between the polymorphism of VDR gene and sensitivity to vitamin D, between the polymorphism of estrogen receptor (hereafter, referred to as ER) gene and sensitivity to estrogen, and between the polymorphism of apolipoprotein E (hereafter, referred to as ApoE) gene and sensitivity to vitamin K2, the VDR genotype AAB (B being a genotype that is not cleaved with Bsm I in the intron region between exon 8 and exon 9) is significantly lower in sensitivity to vitamin D than aabb. Also, the ER genotype PpXx (P and X being genotypes that are not cleaved with Pvu II and Xba I, respectively) is significantly higher in sensitivity to estrogen than other genotype groups, and the ApoE4(+) group is significantly lower in sensitivity to vitamin K2 than the ApoE4(xe2x88x92) group.
Further, it is described that the genotype group whose Restriction Fragment Length Polymorphism (RFLP) pattern obtained by cleaving the vitamin D-binding protein (DBP) gene with Hae III and Sty I is of the GC2-2 type is higher in sensitivity to vitamin D than the other groups [JP-A-8-201373].
However, each of these results is used to anticipate sensitivity to one medicine based on the polymorphism of one gene and enable nothing other than anticipating whether one genotype is higher in sensitivity than other genotypes with respect to one medicine. In other words, it is anticipated that those persons whose VDR genotype is aabb and whose ApoE genotype is ApoE4(xe2x88x92) are higher in sensitivities to vitamin D and vitamin K2, respectively, than those persons who have other VDR and ApoE genotypes. However, it cannot be anticipated which one of vitamin D and vitamin K2 is to be administered to patients having such genotypes in order to obtain a higher therapeutic effect. Therefore, ultimately, to know to which one of different medicines a patient has higher sensitivity, there has been no other way than to administer to the patient one single medicine after another in a span of several years for each medicine and to look at the results. In particular, estrogen not only has a great therapeutic effect but also has a great side effect so that there has been a fear that long-term administration of it to patients could significantly deteriorate their QOL (Quality of Life).
Therefore, an object of the present invention is to provide a means for anticipating to which one of a plurality of remedies for osteoporosis a patient suffering from osteoporosis or a person who has the possibility of acquiring osteoporosis in the future has a higher sensitivity, thereby avoiding progress of the disease caused by long-term administration of a medicine having a low therapeutic effect so that the QOL of patients can be improved.
The present inventors have made intensive research with a view to achieving the above object and as a result they have found that analysis of a human-derived sample containing genome DNA for polymorphisms of the VDR gene, ER gene and ApoE gene makes it possible to anticipate the sensitivity of the human to one of the medicines, vitamin D, estrogen and vitamin K2, that is higher than to the other two medicines based on the combination of the resulting polymorphisms.
That is, they have found a tendency that in the case where a person has both a VDR genotype that is higher in sensitivity to vitamin D than the other genotypes and an ER genotype that is higher in sensitivity to estrogen than the other genotypes, the person is more sensitive to estrogen than vitamin D.
Further, it was found that in the case where a person has an ER genotype that is higher in sensitivity to estrogen than the other genotypes and an ApoE genotype that is higher in sensitivity to vitamin K2 than the other genotypes, the person is more sensitive to estrogen than vitamin K2, and in the case where a person has a VDR genotype that is higher in sensitivity to vitamin D and an ApoE genotype that is higher in sensitivity to vitamin K2, the person is more sensitive to vitamin K2 than vitamin D, thus having arrived at the present invention. In this embodiment, an ApoE genotype is classified into apolipoprotein E4 allele (+) which includes 2/4 3/4 and 4/4, and apolipoprotein E4 allele (xe2x88x92) which includes 2/2, 2/3 and 3/3, and the ApoE genotype that is higher in sensitivity to vitamin K2 refers to the latter group.
Further, the present inventors also have found that in a case where a person has an apolipoprotein E3 allele (+) 3/3 type, the person is more sensitive to vitamin K2 compared with a case where the person has an apolipoprotein E3 allele (xe2x88x92) (which allele includes all but the 3/3 type). Among apolipoprotein E3 alleles, the order of sensitivity of the alleles to vitamin K2 is 3/3 greater than 2/3 or 3/4 greater than 2/2, 2/4 or 4/4.
It has been found that in the case where a person has an ER genotype that is higher in sensitivity to estrogen than the other genotypes and an ApoE genotype that is higher in sensitivity to vitamin K2 than the other genotypes, the person is more sensitive to estrogen than vitamin K2, and in the case where a person has a VDR genotype that is higher in sensitivity to vitamin D and an ApoE genotype that is higher insensitivity to vitamin K2, the person is more sensitive to vitamin K2 than vitamin D, thus having arrived at the present invention. In this embodiment, an ApoE genotype is classified into apolipoprotein E3 allele (+) which includes the 3/3 type, and apolipoprotein E3 allele (xe2x88x92) which includes the 2/2, 2/3, 2/4, 3/4 and 4/4, types and the ApoE genotype that is higher in sensitivity to vitamin K2 refers to the former group.
That is, the embodiments of the present invention are as follows.
1. A method for anticipating sensitivity to a medicine for osteoporosis, characterized by analyzing respective genetic polymorphisms of a vitamin D receptor gene, an estrogen receptor gene, and an apolipoprotein E gene from a genome DNA contained in a sample obtained from a human, and anticipating, based on a combination of the genetic polymorphisms, that the sample is derived from an individual who shows a specific priority of sensitivities to a plurality of remedies for osteoporosis.
The above method is characterized in that the combination of genetic polymorphisms of the vitamin D receptor gene, estrogen receptor gene, and apolipoprotein E gene is one selected from the group consisting of [B(xe2x88x92) X(xe2x88x92) 4(xe2x88x92)], [B(xe2x88x92) X(xe2x88x92) 4(+)], [(B(xe2x88x92) X(+) 4(xe2x88x92)], [B(xe2x88x92) X(+) 4(+)], [B(+) X(xe2x88x92) 4(xe2x88x92)], [B(+) X(xe2x88x92) 4(+)], [B(+) X(+) 4(xe2x88x92)], and [B(+) X(+) 4(+)] (wherein xe2x80x9cBxe2x80x9d represents a vitamin D receptor allele that is not cleaved with Bsm I in an intron region between exon 8 and exon 9, xe2x80x9cXxe2x80x9d represents an estrogen receptor allele that is not cleaved with Xba I in an intron region between exon 1 and exon 2, xe2x80x9c4xe2x80x9d represents an apolipoprotein E4 allele, and (+) and (xe2x88x92) indicate the presence and absence, respectively, of the allele).
The above method is also characterized in that the combination of genetic polymorphisms of the vitamin D receptor gene, estrogen receptor gene, and apolipoprotein E gene is one selected from the group consisting of [B(xe2x88x92) X(xe2x88x92) 3(xe2x88x92)], [B(xe2x88x92) X(xe2x88x92) 3(+)], [B(xe2x88x92) X(+) 3(xe2x88x92)], [B(xe2x88x92) X(+) 3(+)], [B(+) X(xe2x88x92) 3(xe2x88x92)],[B(+) X(xe2x88x92) 3(+)], [B(+) X(+) 3(xe2x88x92)], and [B(+) X(+) 3(+)](wherein xe2x80x9cBxe2x80x9d represents a vitamin D receptor allele that is not cleaved with Bsm I in an intron region between exon 8 and exon 9, xe2x80x9cXxe2x80x9drepresents an estrogen receptor allele that is not cleaved with Xba I in an intron region between exon 1 and exon 2, xe2x80x9c3xe2x80x9d represents an apolipoprotein E3 allele, and 3(+) indicates 3/3 type and 3(xe2x88x92) indicates genotypes other than 3/3 type of the allele).
2. A reagent for simultaneously detecting genetic polymorphism of a vitamin D receptor (hereinafter, VDR) gene, an apolipoprotein E (hereinafter, ApoE) gene, and an estrogen receptor (hereinafter, ER) gene, comprising amplification primers specific to the respective genes of VDR, ApoE and ER and detection probes for detecting a VDR genetic polymorphism, an ApoE genetic polymorphism, and an ER genetic polymorphism, respectively.
In the reagent for simultaneously detecting genetic polymorphism of VDR, ApoE and ER genes as described above, the genetic polymorphism of the VDR gene is a Bsm I restriction enzyme fragment length polymorphism BB, Bb or bb on intron 8 of the VDR gene, the genetic polymorphism of the ApoE gene is a Hha I restriction enzyme fragment length polymorphism 2/2, 2/3, 2/4, 3/3, 3/4 or 4/4 of the ApoE gene, and the genetic polymorphism of the ER gene is a Xba I restriction enzyme fragment length polymorphism XX, Xx or xx on intron 1 of the ER gene.
3. A gene amplification reagent for amplifying VDR, ApoE, and ER genes, comprising amplification primers (1) and (2) specific to the VDR gene, amplification primers (3) and (4), or (5) and (6) specific to the ApoE gene, and amplification primers (7) and (8), or (7) and (9) specific to the ER gene as described below.
Amplification primers specific to the VDR gene:
Amplification primers specific to the ApoE gene:
Amplification primers specific to the ER gene:
4. A reagent for simultaneously detecting genetic polymorphisms that can measure VDR, ApoE and ER genetic polymorphisms, comprising detection probes (10) and (11) for detecting a VDR genetic polymorphism, detection probes (12) and (13) for detecting an ApoE genetic polymorphism, and probes (14) and (15), or (16) and (17) for detecting an ER genetic polymorphism.
Detection probes for detecting a polymorphism of VDR gene:
Detection probes for detecting a polymorphism of ApoE gene:
Detection probes for detecting a polymorphism of ER gene:
5. A reagent for simultaneously detecting genetic polymorphisms that can measure VDR, ApoE and ER genetic polymorphisms, comprising detection probes (10) and. (11) for detecting a VDR genetic polymorphism, and detection probes (12), (13), (18) and (19) for detecting an ApoE genetic polymorphism, and detection probes (14) and (15), or (16) and (17) for detecting an ER genetic polymorphism.
Detection probes for detecting a polymorphism of VDR gene:
Detection probes for detecting a polymorphism of ApoE gene:
Detection probes for detecting a polymorphism of ER gene:
6. A method for selecting a remedy for a bone-associated disease characterized by relating a combination of genetic polymorphism of VDR, ApoE and ER genes detected by the above mentioned method for detecting genetic polymorphisms of VDR, ApoE and ER genes to a remedy for a bone-associated disease.